Project 2 is focused on development of a treatment for insulin-dependent diabetes mellitus based on transplantation of isolated pancreatic islet cells (IPITx). This will be accomplished by induction of immunologic tolerance to IPITx in a preclinical macaque model, which offers a rigorous proof of principle for clinical utility. We will apply a tolerance induction protocol originally developed in the same macaque model for kidney transplantation to IPITx. This application will involve the use of anti-macaque CD3 epsilon directed diphtheria immunotoxin (IT), currently in an iteration constructed with a F(Ab)2 mAb. We have learned that combining IT with an immunosuppressive drug, 15-deoxyspergualin, creates an early environment for tolerance induction which has proven to be robust and practical. Based on these facts, in Aim 1, we propose to first determine the optimum protocol for establishment of IPITx tolerance in cyclosporine A (CsA). The criteria for evaluation will be graft functional, functional islet mass measured metabolically, and immunological changes (cytokine expression, CTLp changes, etc.) After transplantation. We will directly test the tolerance specificity and immunologic competence of the transplanted subjects with challenge donor and 3rd party skin grafts. When the optimized protocol is selected, we will next perform experiments in Aim 2 to determine if the critical problem of islet graft primary non-function can be circumvented through protecting the grafts with transgenic expression of Bcl-2. This gene therapy approach has proven to be very effective in our hands in prolonging macaque islet graft function (as judged by euglycemia) in SCID mice as compared to unmodified or sham-induced islets. We will first optimize the ex vivo transduction of macaque islets and evaluate their performance metabolically and immunologically. The end result should yield a tolerance induction and gene therapy strategy which will prolong graft survival and euglycemia, possibly with the use of fewer islets. Taken together, these findings could translate into effective, economical and efficient treatments of diabetes.